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A flagging disease of western white pine Molnar, Alexander Charles 1954

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A FLAGGING DISEASE OF WESTERN WHITE PINE by ALEXANDER CHARLES MOLNAR  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY in the Faculty of Forestry  We accept this thesis as conforming to the standards required from candidates for the degree of MASTER OF FORESTRY.  Members of +•*»« Facu^TEy'TaT -Forea try  THE UNIVERSITY OF BRITISH COLUMBIA April, 1954  A FLAGGING DISEASE OF WESTEBH WHITE PINE by Alexander Charles Molnar ABSTRACT A disease, causing severe flagging of young western white pine (Pinus monticola Dougl.), was investigated at H i l l Siding and Arrow Park, British Columbia.  The primary symptom of .the disease was  flagging of twigs, resulting from a rapid necrosis of terminal shoots and less commonly from girdling-lesions on 2-year-old growth. A small percentage of the severely flagged trees died. Damaging effects of the disease were confined to trees under 4.0 years of age and to stand forms with a higher than average component of white pine i n the susceptible age class.  Efforts to isolate the causal  fungus yielded 22 fungi i n culture,! only two of which occurred with sufficient frequency to warrant further study, an unknown fungus "B and Pullularia pullulans (deBary) Berkhout. A search B  for fructifications associated with the disease revealed apothecia of a Tympanis. very similar to those of Tympanis pithya (Karst.) Karst., to be associated with 16 per cent of a random sample of 425 flagged twigs.  The pycnidia of the imperfect stage of Tympanis sp.  occurred less commonly. Spore cultures from Tympanis apothecia were different from those of any of the 22 fungi isolated i n tissue culture and i t seemed probable that Tympanis occurred as a pioneer saprophyte on killed twigs.  External signs of Pullularia pullulans  occurred commonly i n the form of erumpent scleuotia, and external hyphae with resting spores.  A scale insect, resembling Matsucoccus  sp., was found at a high infestation level i n one stand and less  commonly i n the remaining stands. Reasons for discounting the insect as a primary causal agent of flagging are outlined. inoculations with Pullularia pullulans  Field  and greenhouse inoculations  with Pullularia pullulans and the unknown fungus "B" produced questionable results with the latter fungus, but indicated Pullularia pullulans to be pathogenic under the conditions imposed by the experiment. four tables.  Results of inoculation experiments are snmmarized i n The historical background and cultural description  of Pullularia pullulans i s outlined.  Experimental and observational  evidence suggests that the fiungus i s one of the causal agents, and probably the primary agent for the flagging.  On the basis of the  limited damage caused by the flagging and the sporadic occurrence of the susceptible stand form, control measures are deemed unwarranted.  ACKNOWLEDGEMENTS The author i s indebted to Dr. D.C. Buckland, Associate Professor of Forest Pathology, University of British Columbia, Vancouver, for direction i n this study; to Dr. R.E. Foster, Officer-in-Charge, Forest Pathology Unit, Victoria, for advice; to Dr. W.R. Haddow, Forest Pathologist, Department of Lands and Forests, Ontario, for kindly examining some of the Pullularia isolates; and to Dr. W.J. Groves, Senior Plant Mycologist, Botany and Plant Pathology Division, Ottawa, for examining Tympanis sp. collections.  TABLE OF CONTENTS Page INTRODUCTION  1  THE DISEASE  2  Symptomatology  2  Distribution  5  Damage  6  ISOLATION OF CAUSAL AGENT  7  Cultural isolations from diseased tissue  7  Search for fructifications associated with flagging  9  Scale inseots  11  PATHOGENICITY TESTS  12  Field inoculations  12  Greenhouse inoculations  16  PULLULARIA PULLULANS  21  Historical background  21  Occurrence i n nature  2U  Cultural characters  26  The imperfect stage  28  The perfect stage  28  DISCUSSION Fungus B n  29 n  29  Tympanis sp.  29  Pullularia pullulans  30  The scale Insect  32  CONCLUSION  33  BIBLIOGRAPHY  34  LIST OF PLATES Page PLATE I Fig. 1.  35 Map showing areas of severe flagging  PLATE II  36  Fig. 2.  Severe flagging of 30-year-old tree  Fig. 3.  Severe flagging of 12-year-old tree  Fig. 4.  Extension of necrosis into branch  PLATE III  37  Fig. 5. Apothecia of Tympanis sp. Fig. 6.  Pycnidia of Tympanis sp.  Fig. 7. Sclerotia of Pullularia pullulans Fig. 8.  External hyphae of Pullularia pullulans  PLATE IV Fig. 9.  38 Flag resulting from natural infection  Fig. 10. Flag following inoculation with Pullularia pullulans Fig. 11. Dieback approaching stem of inoculated seedling. Fig. 12. Seedling dying 2 months after inoculation with Pullularia pullulans PLATE V  39  Fig. 13. Paired cultures of Pullularia pullulans on malt and potato dextrose  agar.  Fig. 14. Early hyphae following yeast-like stage of Pullularia pullulans on malt agar. Fig. 15. Types of spores i n old malt-agar culture of Pullularia pullulans. Fig. 16. Chains of resting spores from dried culture of Pullularia pullulans.  LIST OF TABLES TABLE I:  Host response, following inoculation with Pullularia -pullulartB and check treatment i n f i e l d inoculations  TABLE II:  14  Fungi isolated from twigB, which flagged following inoculation with Pullularia pullulans and check treatment  TABLE III:  17  Host response, following inoculation with Pullularia pullulans. the unknown fungus B and check treatment in greenhouse inoculations  TABLE IV:  19  Fungi isolated from twigs which flagged following inoculation with Pollularia pullulans. fungus B and check treatment  22  A FLAGGING DISEASE OF WESTERN WHITE FINE INTRODUCTION A disease, causing flagging of twigs and branchlets of immature western white pine (Pinus monticola Dougl.), f i r s t came to the author's attention i n 1950 i n the Slocan Valley at H i l l Siding, British Columbia. It was previously noted i n the same area by Dr. D.C. Buckland, during blister rust surveys on white pine i n 1947.  In 1951 the writer found  another area similarly affected 11 miles north of Arrow Park. The two areas constitute entirely unrelated infection centers, being separated by 20 miles of different forest types and Upper Arrow Lake. The cause of the disease and the reasons for i t s locally epiphytotic abundance were not apparent, and while the overall damage caused and the extent of severe flagging was not great, i t posed a potential problem i f the conditions for a high level of infection should become extended. This type of disease becomes particularly important as increasing areas of mature timber are converted into immature stands, which are more subject to damage by diseases affecting stocking, form and growth, than are mature stands. In 1951, the writer, employed by the Forest Pathology Unit, Forest Biology Laboratory, Victoria, B.C., initiated studies to determine the cause of the disease and the factors governing i t s epiphytotic level.  Permission to use some of the results of this study for a  master's Thesis was kindly granted by Dr. J.E. Bier, Associate Chief, Forest Biology Division and Dr. R.E. Foster, Officer-in-Charge, Forest Pathology Unit, 409 Federal Building, Victoria, B.C.  The project, on which this thesis i s based, has not been completed, and the scope of the paper i s confined to the results obtained up to August 1953, with special reference to Pullularia pullulans (deBary) Berkhout, a fungus found closely associated with the flagging disease. THE DISEASE Symptomology The most striking symptom on western white pine of the flagging  1 disease under investigation i s the brick-red flags death of twigs.  produced by the  Dead needles are retained for a few months to a year,  gradually changing to a light brown or straw-colour.  On trees over 15  to 20 f t . high, flagging f i r s t occurs on the lower half of the crown gradually progressing upward. Ultimately, after several years of flagging, the whole crown i s Involved, becoming distinctively"motheaten' i n appearance (Fig. 2).  Such severely flagged trees are obviously  weakened and even the uninfected parts of the crown have shorter than normal, chlorotlc needles. Leader growth i s markedly reduced also. On trees below 20 f t . , flags may occur anywhere on the crown, although, for some undetermined reason, flagging of the leader i s uncommon (Fig. 3). The maximum height at which typical flags were recorded was 34 f t . , and i t appeared significant that mortality was confined to trees under U5 f t . high; the t a l l e r trees apparently have sufficient crown remaining to sustain their vigor.  Thus, the damaging intensity of the disease  1. So called because the brick-red foliage on the killed twigs stands out conspicuously against the normal green of the rest of the crown.  3 was confined to trees below 4.0 years of age* An examination of the flagged twigs revealed that they were killed i n one of two ways: 1, by girdling lesions produced on the second, rarely on the third jinteraodej 2, by rapid neorosis of the terminal shoot, extending sometimes to the previous years growth} Girdling lesions comprised only a small percentage of the total number of similar necrotic lesions produced on the twigs.  A t a l l y of  the lesions on a random sample of 300 twigs showed that only 8.4, per cent of them girdled the twigs to produce flagging. A large number of small cankers on older bark indicated the large number of lesions which had callused over In the past.  Sections of these cankers showed that  relatively few had originated on wood older than two years.  None showed  evidence of activity for more than one season. Lesions were characterized by a sunken, brown, necrotic area, clearly defined against the normal olive-green of healthy young bark. In size they varied from a tiny brown necrotic spot at a fascicle trace to 5 or 6 cm. In length; the larger frequently the result of one or more coalescing.  Small lesions were invariably centered at needle  bundles or bundle scars.  This was probably the point of origin for most  lesions but was not apparent when two to several fascicle traces were included i n a single lesion. In the spring or early summer the edge of the lesions ran smoothly into healthy bark but later i n the season i t was most commonly cracked and frequently raised, Indicating callus formation between healthy and necrotic tissue.  u Flagging^resulting from rapid necrosis involving the whole terminal shoot, apparently originated i n the region of the terminal bud. Extension of necrosis over part or a l l of the second internode occurred commonly, but very rarely onto the third.  Progress of necrosis from a  twig to larger branches was rare (Fig. 4.), but occurred occasionally on severely flagged and obviously weakened branches.  Death of larger  branches resulted from repeated severe flagging of the twigs. It was not always possible to distinguish between the two types of flagging for frequently they resulted from both terminal and internodal infection.  Flagged twigs bearing only local, non-girdling lesions were  common and i t seemed probable that the flagging resulted from terminal Infection. The dieback type of flagging was much more common than those caused by girdling-lesions and comprised from 50 to 85 per cent of the flagging in a l l areas, but the proportions of the two varied. The girdlinglesion type of flagging occurred most commonly i n a young 15 to 25-yearold pine stand near the Slocan Highway. This area was also the most heavily infested by a scale insect to be discussed later. The exact season of the year when flagging occurred was not determined but apparently most occurred between termination of the seasons growth and before bud break i n the spring. This was evidenced by the very rare occurrence of flags with partly developed needles and shoots.  The rare  exception occurred on heavily flagged and weakened branches on which lesions failed to callus but continued development to the point of girdling during the summer. Furthermore, summer and f a l l  examinations  were made each year and fresh flagging was not noted at these seasons.  5 Severely infected seedlings and trees were commonly attacked by secondary invaders such as bark-beetles and Armillaria root-rot.  The  latter was almost invariably present on dead trees of any size. Distribution Flagging injury causing appreciable damage was found only at H i l l Siding and Arrow Park (Fig. 1). In the latter area damage was confined to approximately 100 acres because the susceptible stand type was isolated.  In the H i l l Siding District infection was more  widespread, extending over several hundred acres and two distinct stand types, but severe flagging was patchy throughout, varying with stand composition.  In addition, minor amounts of flagging  continued north of H i l l Siding to approximately half way between Summit and Box Lakes, where the susceptible stand type disappeared. Limited outbreaks of a similar dieback and canker condition were recorded i n other regions of the white pine type, but with negligible attendant damage. There was no indication that the two areas of heavy flagging were spreading. Severe dieback and twig canker was confined to trees under 40 years of age and to stands or portions of stands with a higher than 2 average pine component. The white pine type i n the Interior  wet-belt  forest rarely exceeds 15 to 20 per cent pine i n admixture with other conifers, although small areas, under 1 or 2 acres, of nearly pure pine are not uncommon i n young stands. 2.  The "Interior" refers to a l l areas of British Columbia east of the Cascade Range.  There were two susceptible age groups at H i l l Siding} a 2 5 to 50-year-old stand containing frequent patches of nearly pure pine; and a stand of recent f i r e origin, 15 to 25 years old, which ran heavy to pine throughout.  The latter stand, because of age and origin,  contained a heavy mixture of hardwood brush. The stand at Arrow Park was unique for pine stands i n British Columbia i n that i t averaged over 70 per cent pine. I t was roughly 2-storied, with an open overstory of 3 5 to 50-year-old pine, and a younger age group, seedlings to 25 years of age, i n the openings. Infection here was confined almost entirely to the latter age group. On the basis of height growth of dominant and co-dominant trees, a l l heavily infected stands were of average,or better, site quality for pine. The s o i l was a deep, alluvial, loamy-sand at Arrow Park and of glacial t i l l origin at H i l l Siding, but a l l areas were well drained. The aspect varied with no difference i n disease intensity noted on this basis. The elevation of both areas was close to  2000  ft.  Damage Economic loss resulting from flagging injury was not great. Mortality at H i l l Siding was less than 1 per cent of the stand, although patches of k i l l i n g as high as 2 5 per cent of the stems were not uncommon, with a resultant understocking i n small areas. At Arrow Park mortality was confined to trees under 15 years old, again resulting in some small patches of understocking. Reduction i n growth rate of severely flagged trees, as shown by ring width and leader growth, was marked, but because of the scattered nature of such damage, loss i n yield per acre was probably not high. As leaders were rarely flagged  7 the form of trees, which survive flagging, was not affected. ISOLATION OF CAUSAL AGENT Cultural isolations from diseased tissue In efforts to isolate the causal agent of flagging injury from diseased material, 375 tissue cultures were prepared from fresh lesions, died-back twigs and adjoining, apparently healthy bark and wood tissue. f  Small pieces of inner bark or sapwood were planted i n slants containing 1.25 per cent Difco malt agar and incubated at room temperature. Two methods were tried to secure isolations free from common contaminants.  In the f i r s t of these methods, small pieces of twigs,  from which isolations were to be taken, were dipped i n 95 per cent alcohol for 10 seconds, followed by 30-seconds immersion i n a 1/1000 solution of mercuric chloride, and washed i n several changes of sterile water.  The outer bark or more, depending on the tissue to be isolated,  was then removed with a flame-sterilized scalpel and dissecting needle and a small piece of tissue removed and planted. In the second method, the outer bark or more was removed and the small pieces of tissue to be planted were flamed before being placed on the agar.  Although both methods  gave satisfactory results, most isolations were prepared using the latter, more rapid, method. A record was kept of each isolate as to source and date of planting, by use of a coding system. For screening purposes, cultures were transferred onto paired Petri plates, one containing 2 per cent Difco malt agar and the other 2 per cent Difco potato dextrose agar.  Cultures were then Incubated i n the  dark at 27 degrees C. for six weeks. Measurements of radial growth were  8 taken until the plates were covered*  A descriptive record was maintained  on the appearance of the cultures for the six week period. At the end of six weeks the cultures were screened on the basis of growth rate on the two media and the gross morphological characteri s t i c s of colour, nature of the advancing zone, nature of the mat *wH reaction on the agar, after Nobles ( 1 9 ) . The resulting groups of l i k e cultures were further checked on the basis of microscopic characters* Particular attention was paid to size of hyphae, nature of hyphal branching and special structures, such as, sexual spores, conidiophores, nodular growths, etc*  As separation was quite readily achieved with the  two media mentioned, no special media were prepared to induce spore production* The 2 3 7 uncontamlnated isolates screened i n this manner readily separated into 22 groups*  Of these, two occurred with sufficiently  high frequency to warrant further testing* as Pullularia  p i l l u l ana  One was later identified  (deBary) Berkhout, but the other s t i l l remains  to be identified and w i l l be designated "fungus B" for future reference. Pullularia  pnllnlftwa  comprised  37  per cent of the total isolates  and was isolated at least once from over 9 0 per cent of lesions and dieback twigs cultured*  It occurred most commonly i n inside bark  isolations but was not uncommon In sapwood isolates.  It was recovered  with high frequenoy from living bark tissue when this was taken within 5 mm. of the necrosis.  There was no significant difference between the  numbers of Isolations of this fungus taken from dieback and local lesions.  9 "Fungus B" comprised 28 per cent of the 237 uncontaminated isolates. Its occurrence was much more sporadic than Pullularia and appeared most commonly when the brown-stained sapwood under local lesions was isolated excluding bark tissue.  It was isolated less commonly when both bark  and sap-wood were included, and never occurred when inside bark alone was planted. The remaining 35 per cent of the isolates comprised 20 different fungi, as yet unidentified.  They have, however, been set aside for  future reference. P u l l n l a r i a pullulans and "fungus B" were later used i n pathogenicity tests, to be described. Search for fructifications associated with flagging A thorough search was made at different seasons for external signs of fungi associated with the disease.  Because of the nature of the  disease and the characteristics of the isolates i n culture, i t was considered highly probable that the causal agent may either be an Ascomycete or a member of the Fungi Imperfect!•  If so, the associated  sporocarps would be either ascocarps or imperfect structures bearing conidia.  Four different structures were found associated with the  disease: 1.  apothecia of a species of Tympanis. very similar to Tympanis  Pithya  2.  (Karst.) Karst. (9), (Fig. 5).  small, black, erumpent/ anostiolate pycnidia, very similar to  the imperfect stage of Tympanis spp. (9), (Fig. 6). 3.  sclerotia, erumpent through the bark of flagged twigs,  without associated pycnidia (Fig. 7), similar to sclerotial  10 material found i n old Pullularia pull"l«"s cultures. A.  external hyphae forming mycelial mats, similar to sooty mould,  but consisting of chains of resting spores or "gemmae" i n distinguishable from those produced by Pullularia i n culture (Fig. 8). The tiny, black, stipitate apothecia of Tympanis occurred most commonly i n the f a l l but were found sporadically throughout the spring and summer. Sixteen per cent of a random sample of 425 flagged twigs, examined i n the f a l l of 1953, bore apothecia of Tymnanls. None were found on local lesions of twigs, which had not flagged. The fungus was also found fruiting profusely i n the f a l l on recent slash i n the vicinity of diseased as well as uninfected areas. The pyonidia of Tympanis sp. occurred from early summer to f a l l on flagged twigs; never on local lesions.  They also occurred on slash  associated with Tympanis apothecia. They were found i n the f a l l on over 50 per cent of the flagged field inoculations. Fungus sclerotia, erumpent through the bark of flagged twigs and very similar to those found i n old cultures of Pullularia. were very common on flagged twigs i n nature, and occurred on 33 per cent of inoculated twigs which had flagged. No sporocarp was found associated with sclerotia. Black mats of hyphae, similar to sooty mould, were found i n varying abundance In a l l infected areas.  They were particularly  abundant at Arrow Park In an area where i n a 1/A-acre patch over 90 per cent of the seedlings suffered mortality after severe flagging. Microscopic examination showed these hyphae to be chains of resting spores, very similar to those found In old, dried up, water cultures  11 of Pullularia.  When a portion of mycelium, scraped off twigs which  had been stored a l l winter, was placed on agar, growth typical of Pullularia resulted. Scale insects A scale insect, a species of Matsucocous (16) (Homoptera: Coccoidea: Hilar gar od Ida e) was present at a high population level in the 15 to 25-year-old stand at H i l l Siding and was found i n lesser abundance in the other flagged stands.  The local lesions and attendant flagging  in the former stand were very similar to flagging injury desoribed by McKenzie et a l (17) on yellow pine (Pinus ponderosa var. scopulorum Engelm.), which he attributed primarily to the Prescott scale (Matsucocous vexillorum Morrison). Flagging injury of white pine was at f i r s t thought to be caused by the scale but investigation revealed this to be very unlikely. Severe flagging damage could not be related to areas of high scale population and i n areas severely infested with scale, trees relatively lightly infested were frequently the most heavily flagged. Some of the most severe flagging injury occurred at Arrow Park where the scale population has been extremely light i n the past four years. Branches, which had been severely and repeatedly infested by the scale, produced symptoms not ordinarily found associated with lesions and flagging.  Such branches showed a marked reduction i n internodal  length, had shorter than normal, chlorotic needles, and a marked hypertrophy of bark cortex tissues.  The twigs were covered with a  dark, granular residue of scale cases and the woolly filaments produced by adult females during oviposit!on.  PATHOGENIC H I TESTS It was evident early i n the isolation studies that one fungus, later identified as Pullularia pullulans. was most consistently associated with diseased material.  Field inoculations were initiated  with this fungus i n October,1952,to determine i t s pathogenicity. Potted seedlings were inoculated i n the greenhouse with P. pullulans and the unknown "fungus B" i n March, 1953. Field inoculations A plot of vigorously growing white pine, 5 to 15 years of age and free from natural infection, was inoculated at Arrow Park i n October, 1952.  Inoculum was prepared by growing cultures of Pullularia pullulans.  isolate ACM 52-5-8, on 2 per cent malt agar u n t i l the agar was completely covered with dark mycelium. Agar plus mycelium was applied directly to current years twigs following one of four methods of treatment. Treatment A The needles were removed, without injuring the bark, for 1 i n . , h a l f way along a current years twig, and the stripped part of the twig wiped off with 70 per cent alcohol.  Next, two 1/2 i n . s l i t s , parallel  to the long axis of the twig, were made i n the bark to the cambium. The inoculum was then applied directly to the wound, covered with absorbent cotton moistened with sterile water, and held i n place with cellulose tape. Treatment B The needles were removed as i n treatment A and a small patch 1/U i n . by 1/2 i n . was scraped through the thin epidermis of the bark.  Inoculum was applied to the wound and bound as in treatment A. Treatment 0 Carried out as treatment A, except that needles were removed without further wounding* Thus, fresh needle scars provided the only injury for fungus invasion. Treatment D Treatment was carried out exactly as treatment A and in addition the inoculated trees were 3/A-girdled at the base by exposing a 2-in. band to the cambium. A series of checks were prepared to conform with each treatment, the only difference being that no inoculum was applied. The field inoculations were examined during the following summer in May, July and August and records were maintained on the condition of the inoculated twigs. The cotton and cellulose binding was removed at the first examination. Table I presents a summary of the symptoms manifested by the inoculated and check twigs up to August, 1953. Four types of host response were recorded: 1. tiormal callus formation, with complete absence of necrosis, indicating healing. Healing was not noted until the July examination. As can be seen from Table I none of the inoculated twigs healed without first exhibiting some signs of necrosis. 2. formation of local lesions around the point of inoculation without apparent signs of healing up to the last examination. There was a definite extension of necrosis over the period of observation. A similar condition was noted in natural infections  TABLE I: HOST RESPONSE, FOLLOWING INOCULATION WITH PULLULARIA PULLULANS AND CHECK TREATMENT, IN FIELD INOCULATIONS. Treatment  Total replication  Healthy healed  Per cent of total  Lesion no healing  Per cent of total  Flagged  Per cent of total  Lesion healing  Per cent of total  Inoculations A  16  0  0.0  4  25.0  6  37.0  6  37.0  B  18  0  0.0  2  11.1  16  88.9  0  0.0  C  14  0  0.0  6  42.9  4  28.6  4  28.6  D  32  0  0.0  0  0.0  28  87.5  4  12.5  Total  80  0  0.0  12  15.0  54  67.5  14  17.5  Checks A  12  8  66.7  0  0.0  4  33.3  0  0.0  B  8  6  75.0  0  0.0  2  25.0  0  0.0  C  8  6  75.0  0  0.0  2  25.0  0  0.0  D  17  3  17.5  0  o.p  14  82.5  0  0.0  Total  45  23  51,1  0  o.o  22  48.9  0  0.0  on weakened branches. 3* Flagging. Flagging f i r s t appeared i n May and continued until July but no new flags were noted at the August examination.  The  early flags showed evidence of rapid necrosis above and below the point of inoculation, and before they turned red the needle bases assumed a blue-black colour from invading fungus hyphae* Needles of twigs which flagged after the cotton binding was removed did not discolour In this way. After the needles of flags turned brick-red they could not be distinguished from natural flags (Figs. 9,10), except at the point of wounding* With the exception of two inoculations (treatment B), a l l early flagging was confined to girdled trees of treatment D. However, apart from the early flagging, the latter trees showed no sign of weakening during the summer and put on normal vigorous growth. On a small number of the flagged twigs necrosis progressed from the point of inoculation down to the second intemode by the end of the summer* /+• necrotic lesions followed by healing. A number of the inoculations showed local lesions around the wound during the early part of the summer but healed to form typical annual-cankers by the August examination. Small black pycnidia, very similar to the imperfect stage of Tymp»rds sp., were found on a small number of flagged twigs i n July and on over 50 per cent of them in August* During the course of examinations isolations were prepared from 45 of the flagged inoculations and from the 22 flagged checks. The isolates were taken from the inside bark and sapwood at varying  distances from the point of inoculation, i n the lesion as well as apparently healthy bark immediately i n advance of necrosis*  A summary  of the results of this work i s presented i n Table II. Pullularia pullulans was re-isolated at least once from each of the inoculated twigs and was isolated from 90 per cent of the flagged checks*  None of the cultures under "other fungus isolates" resembled  cultures of "fungus B" or the spore cultures prepared from apothecia of Tympanis. Greenhouse inoculations Healthy seedlings, apparently free from natural infection, were collected at Arrow Park for use i n greenhouse inoculations*  In March,  well before any sign of bud break, the seedlings were inoculated with Pullularia pullulans and "fungus B".  The inoculum was prepared as  for f i e l d inoculations and applied, following two different treatments* Treatment A This treatment was set up exactly as treatment C of the f i e l d inoculations, i.e., needle scars provided the only injury. Treatment B Treatment B corresponded to treatment A of the f i e l d inoculations, fascicle scars and small s l i t s in the bark providing the wound, and in addition water was withheld from the seedlings i n this treatment for one week prior to inoculation. The seedlings used in the inoculations were graded according to size to provide an equal distribution of sizes i n corresponding inoculation series and checks. There was a maximum of two twigs inoculated on the larger seedlings and one twig on the smaller.  TABLE II: FUNGI ISOLATED FROM TWIGS, WHICH FLAGGED FOLLOWING INOCULATION WITH PULLULARIA PULLULANS AND CHECK TREATMENT. Total twigs cultured  Total isolates  P. pullulans isolates No. Per cent  Other fungus isolates No. Per cent  No growth No. Per Cent  I Inoculated twigs  k5  135  Check twigs  22  66  95  70.4  17  12.6  62.1  19  28.8  23  6  17.0  9.1  Total twigs with P. p a l l , No. Per cent  |  ^5  100.0  20  90.9  18 One set of checks were provided to serve for both fungi i n each of the two treatment series. The f i r s t signs of flagging appeared one month after inoculation, when a few of the twigs i n treatment B showed strong chlorosis. The absorbent cotton and cellulose tape binding was removed from a l l treatments at this time and the inoculations were examined. Examinations were repeated at regular intervals during the summer and as shown i n Table III, five types of host response were noted. 1. normal callus formation indicating healthy healing. 2.  local lesions with no sign of healing.  3.  flagging.  4* necrosis followed by healing to form a typical annual canker. 5. death, following flagging. Normal, healthy, callus formation was initiated earlier i n the greenhouse than i n f i e l d inoculations, probably because of earlier cambial activity i n the warm greenhouse. Necrotic lesions, which showed no signs of healing up to the last examination, were generally smaller i n the greenhouse than i n field inoculations.  The necrosis i n 90 per cent of the cases was  confined to the bark tissue, and i n general extended very l i t t l e after the f i r s t month. Two types of flagging were recorded. Early flagging resulting from rapid necrosis distally from the point of inoculation, was almost entirely confined to treatment B seedlings inoculated with Pullularia.  One case of early flagging noted on a treatment A  seedling was a twig inoculated with "fungus B", but i n this case the  TABLE III: HOST RESPONSE, FOLLOWING INOCULATION WITH PULLULARIA PULLULANS, THE UNKNOWN FUNGUS B AND CHECK TREATMENT, IN GREENHOUSE INOCULATIONS.  Treat- Total ment replicat ion  Healthy healed  Per cent of total  Local _ lesion Percent no of healing total  Flagged  Per cent of total  Lesion healing  Per cent Total Per cent of seedlings dead total  Inoculum P. pullulans A B  12 26  1  8.3  6  0  0.0  3  50.0 11.5  0 11  0.0 42.3  12  M.7 46.2  17  0.0 47.1  33.3 64.3  2  16.7  7  0.0  8  28.6  18  27.8  10.0 25.0  0  0.0  7  3  12.5  14  0.0 0.0  5  8  Inoculum Fungus B A  12  1  8.3  5  B  28  0  0.0  2  M.7 7.1  18 Check  A B  10 24  9  90.0  14  58.3  0 1  0.0 4.2  1 6  20 flagging resulted from a necrotic girdle at the point of inoculation. A l l flagging subsequent to the i n i t i a l examination resulted from necrotic girdling with various degrees of longitudinal extension. Necrosis following flagging generally moved downward quite rapidly, at least to the f i r s t node, and i n some cases progressed over several internodes, eventually k i l l i n g the seedling (Figs. 11, 12). Mortality of seedlings following flagging was confined to Inoculated seedlings i n treatment B. Almost 50 per cent of the Pn11uiaria-inoculated and 27 per cent of the fungis B-inoculated seedlings died foilowing flagging. None of the checks or seedlings in the milder treatment A suffered mortality, and, while the seedlings that died were smaller than average, there was an equal number of small seedlings i n a l l treatment groups. However, the flags, which occurred on the treatment B cheoks, were a l l on larger seedlings and i t i s quite conceivable that smaller seedlings may have died following such flagging. With the exception of the higher incidence of early flagging of Pullularla-inoculated twigs there was very l i t t l e difference i n host response between inoculations with the two fungi. Sectioned lesions showed a tendency for a blue-black stain to occur on exposed wood of PnliTilq-ria  inoculations and was absent on fungus B inoculations.  However, only a small number of the inoculations had wood exposed i n this manner, giving very l i t t l e weight to this difference. Sections of the blue-stained wood showed hyphae very similar to the large, dark, closely-septate hyphae found i n Pullularia cultures. This dark mycelium was not found i n microscopic sections of bark and wood inoculated with  'fungus B'^ but the fine hyphae. which were present, could not be differentiated from the fine hyphae of Pullularia. The fine hyphae of the two fungi are very similar i n culture. Cultural experiments set up to recover the inoculated fungi from diseased tissues produced rather poorer results than isolations from field inoculations. Over half of the isolates failed to show any fungus growth i n culture. Fungus B was not recovered i n a single isolate while Pullularia showed up i n both fungus B and check isolates* Table IV summarizes the results of this work.. PULLULARIA PULLULANS The fungus most commonly isolated from diseased material associated with a flagging disease of western white pine was identified as Pullulftria pullulans (deBary) Berkhout. Identification was based on descriptions of the fungus i n the literature ( 2 , 1 2 , 1 3 , 1 5 ) . 3  In  addition, Dr. W.R. Haddow, Dept. of Lands and Forests, Ontario , who has worked with the fungus (12), kindly examined some of the writer's isolates and wrote i n part: "While i t i s hazardous to identify vegetation myoelia, I would not hesitate to say that your fungus i s very close to, i f not identical with, what we have isolated here from red pine. The latter was recognized by Lagerberg as the same as Hormonema dematoides (Lagerberg and He Lin, 1 9 2 7 ) which they instituted i n the well-founded belief that Dematlum pullulans was invalid." Historical background The name Dematium pullulans was given by deBary ( 1 ) to a fungus with septate mycelium from the segments of which ellipsoidal cells 3.  Correspondence with the author.  TABLE IV: FUNGI ISOLATED FROM TWIGS WHICH FLAGGED FOLLOY7ING INOCULATION WITH PULLULARIA PULLULANS AND FUNGUS B, AND CHECK TREATMENT. ^Other Total twigs T o t a l twigs P.-pullulans Fungus" B' fungus with P. with ' isolates isolates No growth isolates •pullulans fungus B Ho.Per cent No.Per cent Ho.Per cent No.Per cent No.Per cent No. Per cent  Total twigs cultured  Total isolates  Pullularia pullulans  26  78  23  29.5  0  o.o  14  17.9  41  52.6  17  65.4  0  0.0  Fungus B  28  6k  k  4.8  0  0  12  14.3  68  80.9  4  14.3  0  0.0  Check  14  kz  17  k0.5  0  0  7  16.7  18  42.8  14  100.0  0  0.0  Inoculum  23 sprouted freely at the ends or at the sides.  After exhausting the  available food supply the hyphae divided transversely into cells which were as long as broad, becoming thick walled, rounded and dark coloured; but when placed on fresh medium, these resting cells or "gemmae", again yielded sprout cells, either directly or from the branched hyphae which developed f i r s t .  DeBary stated that D.  pullulans was most nearly related to Fumago or Pleospora. Following deBary, Loew (17) published a much more extensive account of the fungus and i s frequently accorded joint authorship with deBary. According to the original description (24), Dematium Persoon comprised forms with erect branching oonidiophores bearing chains of conidia laterally.  These structures are entirely lacking i n Dematium  pullulans. and, while the fungus as found i n nature on the usual substrate would be placed i n the Dematiaceae near Tornla Pers., the budding phase belongs In the Pseudosaccharomycetes, along with Torula Turpin and Pseudosaccharomyces Klocker. Accordingly, Berkhout (3) placed the fungus i n a new genus, Pollularia. with i t s distinctive budding habit as i t s primary diagnostic criterion.  Lagerberg et a l (16)  independently instituted Hormonema for the same reason, but believing that Dematium pullulans deBary comprised heterogenous elements, they dismissed i t as invalid for purposes of typification and used Negers description (21) as typical of their genus. They considered their species, Hormonema dematioides. to be closely allied i f not the same as the latter.  Hobak (28) later made the combination Hormonema pullulans.  24 From the above and according to Haddow (12) the synonomy may be given as follows: PnHularia pullulans (deBary) Berkhout, Dissert. Utrecht. 1923. Dematium pullulans deBary. Morph. u. Physiol. Pilze. 1866. Loew. Jahr. Wiss. Bot. 6: 467-477. 1867. Neger, Flora 110: 67-139. 1918. Hormonema dematioldes Lagerberg and Melin. Svenska Skogs. Tldsk. 25* 2. 1927. Hormonema pullulans Robak. Nyt. Mag. for Natur. 71. 1932. In 1928 Bennett (2) reported that he found on wheat straw, associated with Dematium pullulans an ostiolate, perithecoid fungus which he called Anthostomella pullulans.  He found that plantings from the  ascigerous fructification yielded the mycelium of Dematium pullulans i n culture, which i n time produced Immature perithecia.  Some isolates of  Dematium- pullulans. which Bennett secured i n culture, showed distinct morphological and cultural characters but he found that the differenceswere not apparent on microscopic examination, and referred them a l l to Anthostomella pullulans. The foregoing provides only a brief summary of the controversial issue of the identity of this fungus, but comprehensive reviews have been published by Bennett (2), Hoggan (13), Lagerberg (16) and Neger (21). Occurrence i n nature The fungus i s widespread i n nature and has been reported on many substrata but most commonly as an epiphyte.  Pullularia was isolated i n  culture from the Arctic a i r by Pady (23), and P. pullulans was listed  among the fungus flora i n the atmosphere over Kanpur, India, by Rajan (25)* It was isolated as a mould spore from the atmosphere i n London. England (11), and Yerrall (30) found the spores of P. pullulans of common occurrence i n m i l l yards i n the southern United States* Neger (21) found the fungus a common component of sooty mould on the foliage of trees i n Germany. Lagerberg et a l (16) reported Hormonema dematioides (Pullularia pullulans) on live pine needles and as an important blueing fungus i n pine timber.  It has been reported as an airborne fungus  commonly isolated from the galleries of bark beetles by Mathiesen (18) and Rennerfelt (26), but no positive relation to the insect was found. It has been shown to be of considerable economic importance i n Florida, causing deterioration of paint and plastic surfaces, as reported by Goll and Coffey (10), Reynolds (27) and Vicklung and Manowitz (31). Reynolds carried out extensive studies on the fungus. Gadd (9) and Robak (28) report Pullularia pullulans as one of the common moulds of wet pulp and Robak found i t to be one of the most active pulp staining organisms. Brooks and Hansford (A) found i t as a mould on cold storage meat, while C l i f f e r i and Ashford (5) isolated i t from lesions on various parts of the human body, but did not consider i t pathogenic. While  Pullaria  pullulans has generally been reported as a  saprophyte, i t has also been reported as a cause of disease or injury to plants. Bennett (2) reported i t as a weak parasite on etiolated wheat seedlings. Taylor et a l (29) reported i t as the cause of a fruit spot of stored tomatoes i n West Virginia, and English (6,7) reported the fungus causing a decay i n pears and associated with mouldy cherries i n the Pacific Northwest. Furniss (8) found P. pullulans associated with  26 a weevil attacking naturally established Douglas f i r (Pseudotsuga taxifolia (Poir.) Brit.) reproduction and suggested that the fungus aided the larvae to overcome host resistance*  The writer has isolated  i t i n British Columbia not only from the lesions and killed-back twigs of white pine, discussed i n this paper, but also from similar lesions on wester" hemlock (Tsuga heterophylla (Raf.) Sarg.) and yellow pine (Pinus ponderosa Laws. Haddow (12)) i n his work on the needle blight of red pine (Pinus resinosa Ait.) plantations, conducted inoculation experiments with the fungus and showed i t to be pathogenic on red pine needles under certain conditions*  Jump (15) found the fungus to be  associated with forking i n red pine and suggested that a growth substance produced by P. pullulans may be responsible for the condition* Cultural characters The growth of the fungus on 2 per cent Difco malt agar i s quite rapid, covering a center-inoculated 90 mm. Petri dish In 17 to 18 days* Growth for the f i r s t day or two i s milky-mucoid i n appearance, because of the early yeast-like budding, but soon, a fringe of white mycelium appears. The advancing zone i s slightly bayed, 3 to 5 mm* and 10 to 15 mm* i n advance of the submerged mycelium and aerial mycelium, respectively.  After 3 or 4 days, or when the plate i s about 1/4 covered,  the culture becomes greenish-brown to black to the extent of the aerial mycelium (Fig. 13). Soon after the plate i s covered with mycelium the whole culture becomes black with the dark hyphal elements, and has dirty-white to brown, floccose aerial mycelium. the reverse i s strongly bleached.  At this stage  27  Growth on potato dextrose agar i s faster, covering a 90 mm. Petri dish i n 12 to 15. days (Fig. 13).  In addition the milky-mucoid stage  continues longer and the dark hyphal elements appear later.  Old cultures  on either of these two media produce a black, carbonous rind on the surface and frequently raised^sclerotium-like^structures appear. The hyphae are generally simple-septate throughout, except i n the early yeast-like stage when they are f i r s t starting to form (Fig. L4). Three types of hyphal elements are found: fine, hyaline, sparselyseptate hyphae, 2 to 3 microns i n diameter, most common i n the advancing zone but may be found throughout; course, hyaline, relatively thinwalled, more or less closely-septate hyphae, up to 8 microns i n diameter, most conspicuous immediately following the yeast-like stage; and course, brown, thick-walled, closely-septate hyphae 6 to 8 microns and on occasion up to 10 microns i n diameter. These latter hyphae may be found anywhere In the dark zone of the cultures. The most remarkable characteristic of the fungus i s the p r o l i f i c budding of conidia or blastospores directly from the hyphal oells. This condition i s most apparent i n fresh inoculations of the fungus on soft agar.  In older cultures, various sizes and shapes of hyaline  and dark spores are found (Fig. 15). As cultures dry the cells of the dark hyphae round up to form resting spores or "gemmae' (Fig. 16), which are extremely resistant to drought, heat and cold (2, 12, 27).  Old water cultures of the fungus,  dried for several months, grew quite readily when placed on fresh agar.  28 The Imperfect stage There were no organized structures bearing the imperfect stage of Pullularia pullulans found. Sclerotial material similar to that found In old cultures of the fungus were found erumpent through the bark of naturally-Infected and inoculated twigs but there were no sporooarps associated with these. The fungus was also found In the forms of dark hyphae and resting spores, borne externally on twigs. These were verified i n culture to produce growth indistinguishable from P. pullulans. Fresh sterilized twigs of white pine were inoculated i n moisture chambers with mycelium of P. pullulans. After three months the bark and wood were completely Invaded by fungus mycelium, the latter stained a brownish-grey, and sclerotial material, rather loosely plectenchymatous, broke through the bark. There was nothing, resembling a spore-bearing structure, found associated with these. Haddow (12) found an anostiolate pycnidium closely associated with infections of P. pullulans on red pine needles, which he thought was probably the imperfect stage of the fungus. There was no other reference to imperfect sporocarps found i n the literature. The perfect stage The perfect stage of P. pullulans was not found i n nature or produced i n culture. Only one reference to the perfect stage was found i n the literature (2), previously cited under the section "Historical background".  29  DISCUSSION During the course of this investigation 22 fungi were isolated i n culture from diseased material. Of these only two occurred with sufficient consistency to warrant further study, an unidentified fungus designated as "fungus B", and Pullularia pullulans (deBary) Berkhout. Apothecia and the imperfect stage of Tympanis sp. were frequently associated with flagged twigs and a scale insect, Matsucocous sp., was found at a high infestation level in one area and i n lesser abundance i n other flagged areas. The 20 unidentified fungi, isolated i n culture from diseased material with low frequency and sporadic occurrence, cannot be ruled out entirely from association with the disease, but i t seems probable that they are of minor importance. Fungus B Fungus B was isolated frequently but with sporadic occurrence from diseased material.  Inoculation experiments suggested that i t may be  pathogenic under certain conditions, but i t s pathogenicity must remain in doubt for the present, for i t was not recovered from the inoculated host.  It w i l l be necessary to identify this fungus and replicate  pathogenicity tests before i t s association with the flagging disease can be established. Tympanis sp. Observational evidence seems to indicate that Tympanis i s associated with the flagging only as a saprophyte, becoming established very soon after the twigs are k i l l e d .  That i t was never isolated i n  tissue culture cannot yet be explained, for spore cultures of the  fungus grew well on both malt and potato dextrose agar*  Inoculation  experiments with spore cultures are indicated. Pullularia pullulans Experimental and observational evidence suggests that Pullularia pullulans i s one of the causal agents, and probably the primary agent responsible for the flagging injury described.  Evidence i n support  of this conclusion was found i n the high consistency of the fungus  1  association with the disease, i n the fact that i t was frequently isolated from apparently living tissue, i n the results of the inoculation experiments, and i n some of the characteristics of the fungus, both observed by the writer and recorded by others (12, 27). It was the only fungus isolated from diseased material with both a high frequency and consistency.  The frequency of occurrence of the  erumpent sclerotia and externally-borne resting:: spores of the fungus was not surveyed because their identity was only recently established, but they appeared to be common. Pullularia pullulans was isolated not only from necrotic tissue associated with the disease but also from apparently living bark i n advance of necrosis.  The a b i l i t y of the fungus to invade a living  host has been noted by other investigators (2, 12, 15). Inoculation experiments indicated that P. pullulans. isolated from diseased material associated with flagging injury of white pine, was pathogenic, at least under the conditions imposed by the experiment.  This may be questioned on the basis that a large number of  the check twigs had flagged. It i s not possible, however, to guard against the infection of checks from an outside source with any degree  31 of assurance, particularly i n f i e l d inoculations.  Swabbing the point  of wounding with alcohol and covering i t with cotton and cellulose tape w i l l generally keep infective spores away from the wound; but i t cannot prevent the mycelium of an aggressive fungus, adapted to'both saprogenic and pathogenic habit, from passing under the cotton to infect the wound^ particularly when the conditions under the cotton binding provide an exoellent environment for fungus growth. There were reasons to believe that the checks of both f i e l d and greenhouse inoculations, which showed symptoms similar to those exhibited by inoculated twigs, were infected by P. pullulans from an outside source. The check treatments were set up f i r s t i n each case, thus, inoculum used for the inoculated twigs could not have been the source of Infection.  Pullularia pullulans was isolated from 90 and  100 per cent of the flagged checks i n the field and greenhouse,experiments, respectively.  There were fewer infected checks i n the greenhouse  than i n f i e l d inoculations, presumably because the source of outside inoculum was more limited.  The fungus was found i n nature i n the form  of dark mycelium and resting spores. In this form the fungus has been shown to be highly resistant to the extremes of climate (12, 27). Such resting spores were readily activated i n water culture.  Presumably a  similar environment i s periodically provided i n nature i n the form of rain. The method by which P. pullulans. and other fungi that may be associated with the condition, gain entry to the host was not found. Entrance through uninjured bark or needles i s unlikely.  The scale  insect previously mentioned, however, could quite conceivably provide  32 entrance courts for infection. On yellow pine a related insect species was considered the primary agent causing flagging injury (20); and other species of Matsucocous are credited with a b i l i t y to damage bark tissues (19).  However, this aspect of the study w i l l require further  attention before conclusive evidence on the relation of the scale to injury on white pine can be provided. The scale insect The reasons for discounting the Matsucocous as a primary agent causing flagging were outlined under "ISOLATION OF CAUSAL AGENT", but w i l l be reiterated briefly.  There was a lack of correlation between the  intensity of flagging and intensity of scale infestation. There were symptoms found associated with repeated severe scale infestations not ordinarily found associated with flagging.  There was extension of  necrosis i n the dieback type of flags to areas obviously remote from the local influence of the immobile feeding stage of the scale.  In regard  to this latter point, the possibility of toxic substances, secreted by the scale, causing necrosis remote from the point of feeding cannot be rejected without evidence. However, on the basis of the other points of evidence, i t seems probable that i f the scale insect i s associated with the flagging, i t s role i s confined to providing entry for fungus invasion; and since there was a lack of correlation between intensity of scale infestation and severity of flagging, factors other than incidence of entrance courts limit the frequency of successful fungus invasion.  An example of such factors may be the need for suitable  conditions of humidity and temperature to coincide with a particular stage of feeding injury.  33 Conclusion The overall damage caused by the flagging injury under investigation and the extent of the infected area, based on the past four years observation, did not appear to be increasing.  Damaging Intensity of the  disease appears to be confined to trees under 40 years of age and to stands with a higher than average component of pine within the susceptible age class* On the basis of the foregoing, control measures are not indicated, particularly since the susceptible stand form i s of sporadic occurrence* Experimental and observational evidence seems to Indicate that P. pullulans i s the primary agent responsible for the flagging injury and that other fungi may be involved.  However, the scope of the  investigation has been limited to date and replication of pathogenicity tests and further cultural studies are indicated.  Studies on the  histology of the disease and an evaluation of the factors which Limit the activity of the fungus seem desirable*  BIBLIOGRAPHY 1*  Bary de, A.  Vergleichende Morphologie der Pllze, Mycetozoen  und Bacterien. I884. 2.  Bennett, F.T*  On Dematium pullulans deB. and i t s aseigerous stage  Ann. Appl. B i o l . 15: 371-391. 2 p i . , 5 figs. 3.  Berkhout, Kristine M.  1928.  De Schimraelgeschlachten. Monilia, Oidium,  Oospora, en Torula. Dissert. 1-71. 4 pl» Univ. Utrecht. 1923. (R.A.M. 3: 4*  555).  Brooks, F.T. and C.G. Hansford.  Mould growths upon cold-storage  meat. Trans. Brit. Myc. Soc. 8: 113-142. 5.  C l i f e r r i , R. and B.K. Ashford.  1923.  Two strains of Pullularia pullulans  (deBary) Berkhout isolated from the human skin. J . of Publ. Health and Trop. Med. 5: 188-195. 6.  English, W.H.  The Porto Rico 1929.  Fungi isolated from mouldy cherries i n the Pacific  Northwest. Proc. Amer. Soc. Hort. Sci. 4.0: 172-176. 532).  (R.A.M. 21: 7.  English, W.H.  1943.  Taxonomic and pathogenicity studies of the fungi  which cause decay of pears i n Washington. Res. Stud. C o l l . Wash. 8: 127-128. 1940 8.  Furniss, R.L.  (R.A.M. 21:  531).  Biology of Cylindrocopturus furnissi Buchanan on  Douglas-fir. J . Econ. Ent. 35r 853-859. 1 Fig. 9*  Gadd, G.O.  1942.  On the microbiological problems of the pulp and paper  industries.  Paperi fa Puu (Papers and Timber) 33: 49-52.  1951*  (R.A.M. 31: 3. 1952). 10.  Goll, M. and G. Coffey. Mildew of paint surfaces. Paint O i l Chem. Rev. 3 pp. 11 f i g s . Aug. 1948.  (R.A.M. 30: 202. 1951).  35 11. Groves, J . Walton and A. Mavis Leach. The species of Tympanis occurring on Pinus. Mycologia, 41: 59-76. 194.9. 12.  Haddow, W.R.  Needle blight and late f a l l browning of red pine  (Pinus resinosa Ait.)  caused by the gall midge (Ceoidomyldae)  and the fungus Pullularia pullulans (deBary) Berkhout. Trans. Roy. Canad. Inst. 28, Pt. 2: 161-189. 1941. 13. Eoggan, Isme, A.  On Dematium pullulans deBary.  Trans. B r i t . Mya.  Soc. 9: 100-107. 1924. 14*  Hyde, H.A. and D.A. Williams. A census of the mould spores i n the atmosphere. Nature. Lond. 164: 4.172, pp. 668-669. 194-9 (R.A.M. 29: 4 0 . 1950).  15. Jump, J.A.  A study of forking i n red pine.  Phytopath. 28: 798-811.  1938. 16.  Lagerburg, T., G. Lundberg, and E. Melin.  Biologioal and practical  researches into the blueing i n pine and spruce. Svenska Skogs. Tidsr. 25: 145-272. 1927. 17.  Loew, E.  Ueber Dematium pullulans deBary. Jahr. f • Wiss. Bot.  6: 467-477. 1867-68. 18.  Mathiesen, Aino. Uber unige mit Barkenkafern associierte Blauepilze in Sohweden. Oikos 2: 275-308. 1950.  19. Mckenzie, Howard L.  The seasonal history of Matsucocous vexillorum  Morrison (Homoptera: Goccoidea: Margarodidae). Microentomology 8: 42-52, i l l u s . 1943. 20.  Mckenzie, H.L., L.S. G i l l , and Don E. E l l i s .  The Prescott scale  (Matsuccocus vexillorum) and associated organisms that cause flagging injury to ponderosa pine i n the southwest. J. Agri.  36 Ees. 76: 33-51. 1948.  21«  Neger, F.W. Experimental!* Untersuehungen TJber Rustaupilze. Flora. UO: 67-139. 1918.  22. Nobles, M.K. Studies i n forest pathology, V.I. Identification of cultures of wood-rotting fungi. Can. J. Research, C. 26: 281-431. 1948.  23.  Fady, S.M. Fungi isolated from Arctic a i r i n 1947. Canad. J. Bot. 29: 46-56, 1 p i . 1951. (R.A.M. 31: 504. 1948).  24. Persoon, C.H. Tentamen dispositionis Methodlcae fungorum 41* 1797. 25. Rajan, B.S.V., S.S. Nigam, and R.E. Shukla. A study of the atmospheric fungal flora of Kanpur. Proc. Ind. Acad. Sci., Sect. B. 35: 33-37, 1 graph. 1952. (R.A.M. 31: 567, 1952). 26. Rennerfelt, Erik. TJber den zusammenhangzwischen dem verblauen des Holzes und den Insecten. Oikos 2: 120-137. 1950. (R.A.M. 31: 42. 1948). 27. Reynolds, E.S. Pullularia as a cause of deterioration of paint and plastic surfaces i n South Florida. Mycologia. 42: 432-448* 2 Figs. 1950. 28. Robak, H. InvestigaHons regarding fungi i n Norwegian ground wood pulp and fungal infection at wood pulp mills. Nyt. Mag. for Naturvidenskaberne. 71. 185-330. 1932. 29. Taylor, Carlton E. and Leland Shanor. Pullularia pullulans storage fruit spot of tomato. Phytopath. 35: 210-212. 1 Fig. 1945. 30. Verrall, A.F. Relative importance and seasonal prevalence of some wood staining fungi i n the Southern United States. Phytopath. 29: 1031-10J1. 1939.  37 31.  Vichlung, R.E. and M. Manowitz. Mildewing of paints and i t s prevention.  Paint Varn. Prod. 31: 8-11, 26, 4 Figs. 1951.  (R.A.M. 31: 136. 1952.  PLATE  I  Fig. 1. Map showing areas of severe flagging.  PLATE II Fig. 2 .  Severely flagged 30-year-old white pine.  Fig. 3 . Severely flagged 12-year-old understory white pine. Fig. U*  Showing extension of necrosis from flagged twig into larger branch.  PLATE III Fig. 5.  Apothecia of Tympanls sp. on flagged white pine twig, (x 18).  Fig. 6.  Pycnidia of Tympanis sp. dead white pine twig.  Fig. 7.  (Approx X 20)  Erumpent sclerotia of Pullularia pullulans on flagged twig of the dieback type.  Fig. 8.  (Approx X 16).  External hyphae of Pullularia pullulans on dead twig.  (Approx X 3).  PLATE  Fig. 9.  IV  Flag of the dieback type resulting from natural infection. ( X f ) .  Fig. 10. Flag produced following f i e l d inoculation of white pine twig with Pullularia pullulans. partly defoliated.  ( X f).  Fig. 11. Symptoms on inoculated seedling showing necrosis approaching node.  ( X /+).  Fig. 12. Seedling dying following inoculation with Pullularia pullulans. ( X  PLATE  V  Fig. 13. Paired plates of Pullularia pullulans . Upper on potato dextrose agar, lower on malt agar.  (Approx. X £ ) .  Fig. L4. Fine hyphae of P. pullulans following yeastlike stage. Note blastospores.  (Approx. X 400).  Fig. 15. Various hyaline and dark spore from old P. pullulans culture.  Note cells of hyphae rounding up to form  resting spores.  (Approx. X 300).  Fig. 16. Dark hyphae and chains of resting spores from old, dried up P. pullulans culture.  (Approx. X 300).  


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